Ovine CRF (oCRF) was characterized in 1981 as a 41-residue amidated peptide. oCRF lowers blood pressure in mammals when injected peripherally and stimulates the secretion of ACTH and β-endorphin. Rat CRF (rCRF) was later isolated, purified and characterized; it was found to be a homologous, amidated hentetracontapeptide as described in U.S. Pat. No. 4,489,163, the disclosure of which is incorporated herein by reference. The amino acid sequence of human CRF (hCRF) has now been determined to be the same as that of rCRF.
In about 1981, a 40-residue amidated peptide was isolated from the skin of the South American frog Phyllomedusa sauvagei and referred to as sauvagine. It was characterized by Erspamer et al. and was described in Regulatory Peptides, Vol. 2 (1981), pp. 1–13. When given intravenously (iv), sauvagine and oCRF have been reported to cause vasodilation of the mesenteric arteries so as to lower blood pressure in mammals and also in stimulating the secretion of ACTH and β-endorphin. However, when administered intracerebroventricularly (icv), there is an elevation of heart rate and mean arterial blood pressure, which are secondary to activation of the sympathetic nervous system.
Although originally isolated and characterized on the basis of its role in this hypothalamopituitary-adrenal (HPA) axis, CRF has been found to be distributed broadly throughout the central nervous system as well as in extraneural tissues, such as the adrenal glands, placenta and testes, where it may also act as a paracrine regulator or a neurotransmitter. Moreover, the likely involvement of CRF in affective disorders, such as anxiety, depression, alcoholism and anorexia nervosa, and in modulating reproduction and immune responses suggests that changes in CRF expression may have important physiological and pathophysiological consequences. For example, perturbations in the regulatory loops comprising the HPA axis often produce chronically elevated levels of circulating glucocorticoids; such patients display the physical hallmarks of Cushing's syndrome, including truncal obesity, muscle-wasting, and reduced fertility.
In addition to its role in mediating activation of the hypothalamic-pituitary-adrenal, CRF has also been shown to modulate autonomic and behavioral changes, some of which occur during the stress response. Many of these behavioral changes have been shown to occur independently of HPA activation in that they are not duplicated by dexamethasone treatment and are insensitive to hypophysectomy. In addition, direct infusion of CRF into the CNS mimics autonomic and behavioral responses to a variety of stressors. Because peripheral administration of CRF or a CRF analog fails to affect certain of these changes, it appears that CRF exhibits a direct brain action with respect to such functions, which include appetite suppression, increased arousal and learning ability. However, CRF antagonists given peripherally attenuate stress-mediated increases in ACTH secretion, and when delivered into the cerebral ventricles can mitigate stress-induced changes in autonomic activity and behavior.
As a result of the extensive anatomical distribution and multiple biological actions of CRF, this regulatory peptide is believed to be involved in the regulation of numerous biological processes. CRF has also been implicated in the regulation of inflammatory responses. Although it has been observed that CRF plays a pro-inflammatory role in certain animal models, CRF appears to suppress inflammation in others by reducing injury-induced increases in vascular permeability.
CRF analogs are generally effective in the prophylaxis and/or treatment of stress-related illnesses, mood disorders such as depression, major depressive disorder, single episode depression, recurrent depression, child abuse induced depression, postpartum depression, dysthemia, bipolar disorders and cyclothymia; chronic fatigue syndrome; eating disorders such as anorexia and bulimia nervosa; generalized anxiety disorder; panic disorder; phobias; obsessive-compulsive disorder, post-traumatic stress disorder, pain perception such as fibromyalgia; headache; gastrointestinal diseases; hemorrhagic stress; ulcers; stress-induced psychotic episodes; fever; diarrhea; post-operative ileus, colonic hypersensitivity; irritable bowel syndrome; Crohn's disease; spastic colon; inflammatory disorders such as rheumatoid arthritis and osteoarthritis; pain; asthma; psoriasis; allergies; osteoporosis; premature birth; hypertension, congestive heart failure; sleep disorders; neurodegenerative diseases such as Alzheimer's disease, senile dementia of the Alzheimer's type, multiinfarct dementia, Parkinson's disease, and Huntington's disease; head trauma; ischemic neuronal damage; excitotoxic neuronal damage; epilepsy; stroke; spinal cord trauma; psychosocial dwarfism; euthyroid sick syndrome; syndrome of inappropriate antidiarrhetic hormone; obesity; chemical dependencies and addictions; drug and alcohol withdrawal symptoms; cancer; infertility; muscular spasms; urinary incontinence; hypoglycemia and immune dysfunctions including stress induced immune dysfunctions, immune suppression and human immunodeficiency virus infections; and stress-induced infections in humans and animals.
CRF antagonists containing D-isomers of α-amino acids were developed, such as those shown in U.S. Pat. No. 5,109,111. Antagonists of CRF are disclosed in U.S. Pat. No. 4,605,642, issued Aug. 12, 1986, the disclosure of which is incorporated herein by reference. Cyclic CRF antagonists exhibiting biopotency were later developed as disclosed in U.S. Pat. Nos. 5,493,006, 5,510,458, 5,777,073 and 5,874,227.
Recent clinical data have implicated corticotropin-releasing factor (“CRF”) in neuropsychiatric disorders and in neurodegenerative diseases, such as Alzeimer's disease. Alzheimer's disease is a neurodegenerative brain disorder which leads to progressive memory loss and dementia. By current estimates, over two million individuals in the United States suffer from this disease. In particular, several lines of evidence have implicated CRF in Alzheimer's disease (AD) (Behan et al., Nature 378(16):284, 1995). First, there are dramatic (greater than 50%) decreases in CRF (Bissette et al., JAMA 254:3067, 1985; DeSouza et al., Brain Research 397:401, 1986; Whitehouse et al., Neurology 37:905, 1987; DeSouza, Hospital Practice 23:59, 1988; Nemeroff et al., Regul. Peptides 25:123, 1989) and reciprocal increases in CRF receptors (DeSouza et al., 1986; DeSouza, 1988) in cerebrocortical areas that are affected in AD, while neither CRF nor CRF receptors are quantitatively changed in non-affected areas of the cortex (DeSouza et al., 1986). Second, chemical affinity crosslinking studies indicate that the increased CRF receptor population in cerebral cortex in AD have normal biochemical properties (Grigoriadis et al., Neuropharmacology 28:761, 1989). Additionally, observations of decreased concentrations of CRF in the cerebrospinal fluid (Mouradian et al., Neural Peptides 8:393, 1986; May et al., Neurology 37:535, 1987) are significantly correlated with the global neuropsychological impairment ratings, suggesting that greater cognitive impairment is associated with lower CRF concentrations in cerebrospinal fluid (Pomara et al., Biological Psychiatry 6:500, 1989).
Available therapies for the treatment of dementia are severely limited. Tacrine™, a recently approved drug, leads to only marginal memory improvement in Alzheimer's patients, and has the undesirable side effect of elevating liver enzymes. Alterations in brain CRF content have also been found in Parkinson's disease and progressive supranuclear palsy, neurological disorders that share certain clinical and pathological features with AD. In cases of Parkinson's disease, CRF content is decreased and shows a staining pattern similar to cases of AD (Whitehouse et al., 1987; DeSouza, 1988). In progressive supranuclear palsy, CRF is decreased to approximately 50% of control values in frontal, temporal, and occipital lobes (Whitehouse et al., 1987; DeSouza, 1988).
Some depressive disorders are also associated with decreased levels of CRF. Patients in the depressive state of seasonal depression and in the period of fatigue in chronic fatigue syndrome demonstrate lower levels of CRF in the cerebrospinal fluid (Vanderpool et al., J. Clin. Endocrinol. Metab. 73:1224, 1991). Although some depressions have a high improvement rate and many are eventually self-limiting, there are major differences in the rate at which patients recover. A major goal of therapy is to decrease the intensity of symptoms and hasten the rate of recovery for this type of depression, as well as preventing relapse and recurrence. Anti-depressants are typically administered, but severe side effects may result (e.g., suicidality with fluoxetine, convulsions with bupropion). (See Klerman et al. in Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, R. F. Prien and D. S. Robinson (eds.), Raven Press, Ltd. N.Y., 1994, p. 281.)
Hypoactivation of the stress system as manifested by low CRF levels may play a role in other disorders as well. For examples, some forms of obesity are characterized by a hypoactive hypothalamic-pituitary-adrenal axis (Kopelman et al., Clin. Endocrinol (Oxford) 28:15, 1988; Bernini et al., Horm. Res. 31:133, 1989), some patients with post-traumatic stress syndrome have low cortisol excretion (Mason et al., J. Neu. Men. Dis. 174:145, 1986), and patients undergoing withdrawal from smoking have decreased excretion of adrenaline and noradrenaline, as well as decreased amounts of cortisol in blood (West et al., Psychopharmacology 84:141, 1984; Puddy et al., Clin. Exp. Pharmacol. Physiol. 11:423, 1984). These manifestations all point to a central role for CRF in these disorders because CRF is the major regulator of the hypothalamic-pituitary-adrenal axis. Treatments for these disorders have poor efficacy. For example, the most effective approach to treatment of obesity is a behavior-change program. However, few participants reach goal weight and the relapse rate is high (see Halmi et al. in Clinical Evaluation of Psychotropic Drugs: Principles and Guidelines, R. F. Prien and D. S. Robinson (eds.), Raven Press, Ltd. New York, 1994, p. 547).
In view of the deficiencies in treatments for such disorders and diseases, more effective treatments are needed. The present invention exploits the correlation of reduced levels of CRF with various neuro-physiologically based disorders and diseases to effectively treat such diseases by increasing levels of free CRF, and further provides other related advantages. Because these actions are mediated by CRFR2, CRFR2-selective analogs are preferred over non-selective analogs due to the possible side effects resulting from activation of other CRF receptors.
The physiological actions of CRF are mediated through activation of at least two high affinity receptors, CRFR1 and CRFR2, which are members of the seven-transmembrane family of receptors [Chen R., et al, P.N.A.S., 90:8967–8971 (1993), Perrin, M., et al., P.N.A.S, 92:2969–2973 (1995), Lovenberg, T., et al., P.N.A.S., 92:836–840 (1995), K. D. Dieterich et al. Exp. Clin. Endocrinol. Diabetes (1997) 105:65–82 and J. Spiess et al. Trends Endocrinol. Metab. (1998) 9:140–145]. Evidence from transgenic knockouts [A. Contarino et al., Brain Res. (1999) 835:1–9, G. W. Smith et al., Neuron (1998) 20:1093–1102 and P. Timpl et al., Nature Genet. (1998) 19:162–166], antisense oligonucleotide studies [S. C. Heinrichs et al., Regul. Pept. (1997) 71:15–21, G. Liebsch et al., J. Psychiatric Res. (1999) 33:153–163 and T. Skutella et al., Neuroscience (1998) 85: 795–805] and CRFR1 receptor antagonists [K. E. Habib et al., Proc. Natl. Acad. Sci. USA (2000) 97:6079–6084., J. Lundkvist et al., Eur. J. Pharmacol. (1996) 309:195–200., R. S. Mansbach et al., Eur. J. Pharmacol. (1997) 323:21–26 and S.C. Weninger et al., Proc. Natl. Acad. Sci. USA (1999) 96:8283–8288] provide evidence for the involvement of CRFR1 receptors in mediating the anxiogenic effects of CRF.
A human CRF receptor was the first to be reported, and it was cloned from a human Cushing pituitary tumor as described in Chen R., et al, P.N.A.S., 90, 8967–8971 (October 1993). It is referred to as hCRFR1 or hCRF-RA and has 415 amino acids; a splice variant thereof includes an insert of 29-amino acids. A rat CRF receptor was isolated, approximately contemporaneously, by hybridization from a rat brain cDNA library. It is referred to as rCRFR1; it also has a 415 amino acid sequence and was disclosed in Perrin, M., et al., Endocrinology, 133, 3058–3061 (1993). It was found to be 97% identical at the amino acid level to the human CRFR1, differing by only 12 amino acids. The receptor has since been reported to be widely distributed throughout the brain and the pituitary and to be likely present in the adrenals and spleen. CRF(1) receptors (CRFR1) have been relatively well characterized, and antagonists to this receptor effectively block stress-induced behaviors in rodents.
The CRFR2 receptor was identified more recently [T. Kishimoto et al., Proc. Natl. Acad. Sci. USA (1995) 92:1108–1112, W. A. Kostich et al., Mol. Endocrinol. (1998) 12:1077–1085, T. W. Lovenberg et al., Proc. Natl. Acad. Sci. USA (1995) 92:836–840. and M. Perrin et al., Proc. Natl. Acad. Sci. USA (1995) 92:2969–2973.] and exists as at least three splice variants. CRFR1 and CRFR2 receptor subtypes are 70% homologous in their amino acid sequences but appear to be pharmacologically [D. P. Behan et al., Mol. Psychiatry (1996) 1:265–277. and K. D. Dieterich et al., Exp. Clin. Endocrinol. Diabetes (1997) 105:65–82.] and anatomically distinct [D. T. Chalmers et al., J. Neurosci. (1995) 15:6340–6350. and D. H. Rominger et al., J. Pharmacol. Exp. Ther. (1998) 286:459–468]. The CRFR2 isoform is highly expressed in limbic brain regions and in the hypothalamus, suggesting possible roles in anxiety, depression and appetite control. The abundance of CRFR2 protein and mRNA in the lateral septum [D. T. Chalmers et al., J. Neurosci. (1995) 15:6340–6350 and D. H. Rominger et al., J. Pharmacol. Exp. Ther. (1998) 286:459–468], and the central role of septum in limbic brain circuitry, mediating emotional responses such as fear, anxiety and aggression [J. Menard and D. Treit, Physiol. Behav. (1996) 60:845–853. and E. Yadin et al., Physiol. Behav. (1993) 53:1077–1083].
CRFR1 is distributed throughout the brain and the sensory and motor relay sites, whereas CRFR2 is expressed in regions of the body where there is little or no expression of CRFR1, such as peripheral sites, e.g. the blood vessels, the heart, the GI tract, the lungs and the skin. In addition, while CRFR1 expression is very high in neocortical, cerebellar, and sensory relay structures, CRFR2 expression is generally confined to subcortical structures. The highest levels of CRFR2 mRNA in brain are evident within the lateral septal nucleus, the ventromedial hypothalamic nucleus and the choroid plexus. Moderate levels of CRFR2 expression are also evident in the olfactory bulb, amygdaloid nuclei, the paraventricular and supraoptic nuclei of the hypothalamus, the inferior colliculus and 5-HT-associated raphe nuclei of the midbrain. CRFR2-expressing cells are also evident in the bed nucleus of the stria terminalis, the hippocampal formation and anterior and lateral hypothalamic areas. In addition, CRFR2 receptor mRNA is also found in cerebral arterioles throughout the brain. Within the pituitary gland, CRFR2 mRNA is detectable at low levels in scattered cells while CRFR1 mRNA is readily detectable in anterior and intermediate lobes.
This heterogeneous distribution of CRFR1 and CRFR2 mRNA suggests distinctive functional roles for each receptor in CRF-related systems. The CRFR1 may be regarded as the primary neuroendocrine pituitary CRF receptor and important in cortical, cerebellar and sensory roles of CRF. The anatomical distribution of CRFR2 mRNA indicates a role for this novel receptor in hypothalamic neuroendocrine, autonomic and general behavioral actions of central CRF. The location of CRFR2, which are highly expressed in limbic brain regions, supports the involvement of these receptors in fear-conditioning (Ho et al., Brain Res Mol Brain Res (2001) 89(1–2):29–40). Additional studies to determine the effects of simultaneous inhibition of both receptor subtypes show that rats receiving both CRFR2 antisense oligonucleotide and CRFR1 antagonist froze significantly less than animals treated with either agent alone. These results provide additional evidence for the role of CRFR2 in mediating the stress-induced actions of endogenous CRF. CRFR2 is also involved in central autonomic and appetitive control. CRFR2 exists as three splice variants of the same gene and have been designated CRFR2a, CRFR2b and CRFR2g. CRFR2b is used interchangeably with CRFR2β. The pharmacology and localization of all of these proteins in brain has been well established. The CRFR1 subtype is localized primarily to cortical and cerebellar regions while the CRFR2a is localized to subcortical regions including the lateral septum, and paraventricular and ventromedial nuclei of the hypothalamus. The CRFR2b is primarily localized to heart, skeletal muscle and in the brain, to cerebral arterioles and choroid plexus. The CRFR2g has most recently been identified in human amygdala.
Both CRFR1 and CRFR2 were found in the pituitary and throughout the neocortex (especially, in prefrontal, cingulate, striate, and insular cortices), amygdala, and hippocampal formation of primates. In primates, both CRFR1 and CRFR2 may be involved in mediating the effects of CRF on cognition, behavior, and pituitary-adrenal function. The presence of CRFR1 (but not CRFR2) within the locus coeruleus, cerebellar cortex, nucleus of the solitary tract, thalamus, and striatum and of CRFR2 (but not CRFR1) in the choroid plexus, certain hypothalamic nuclei, the nucleus prepositus, and the nucleus of the stria terminalis suggests that each receptor subtype also may have distinct functional roles within the primate central nervous system. See, e.g., Sanchez et al., J. Comp. Neurol. 408:365–377.
CRF has been widely implicated as playing a major role in modulating the endocrine, autonomic, behavioral and immune responses to stress. The recent cloning of multiple receptors for CRF as well as the discovery of non-peptide receptor antagonists for CRF receptors have begun a new era of CRF study. Presently, there are five distinct targets for CRF with unique cDNA sequences, pharmacology and localization. These fall into three distinct classes, encoded by three different genes and have been termed CRFR1 and CRFR2 (belonging to the superfamily of G-protein coupled receptors) and CRF-binding protein. Expression of these receptors in mammalian cell lines has made possible the identification of non-peptide, high affinity, selective receptor antagonists. While the natural mammalian ligands oCRF and r/hCRF have high affinity for the CRFR1 subtype, they have lower affinity for the CRFR2 family making them ineffective labels for CRFR2. [125I]Sauvagine has been characterized as a high affinity ligand for both CRFR1 and the CRFR2 subtypes and has been used in both radioligand binding and receptor autoradiographic studies as a tool to aid in the discovery of selective small molecule receptor antagonists. A number of non-peptide CRFR1 antagonists that can specifically and selectively block the CRFR1 receptor subtype have recently been identified. Compounds such as CP 154,526, NBI 27914 and Antalarmin inhibit CRF-stimulation of cAMP or CRF-stimulated ACTH release from cultured rat anterior pituitary cells. Furthermore, when administered peripherally, these compounds compete for ex vivo [125I]sauvagine binding to CRFR1 in brain sections demonstrating their ability to cross the blood-brain-barrier. In in vivo studies, peripheral administration of these compounds attenuate stress-induced elevations in plasma ACTH levels in rats demonstrating that CRFR1 can be blocked in the periphery. Furthermore, peripherally administered CRFR1 antagonists have also been demonstrated to inhibit CRF-induced seizure activity. These data clearly demonstrate that non-peptide CRFR1 antagonists, when administered systemically, can specifically block central CRFR1 and provide tools that can be used to determine the role of CRFR1 in various neuropsychiatric and neurodegenerative disorders. In addition, these molecules will prove useful in the discovery and development of potential orally active therapeutics for these disorders. McCarthy et al., Curr Pharm Des. (1999) 5(5):289–315.
Because the CRFR1 control different functions than the CRFR2, it would be valuable to be able to regulate one family of receptors without significantly affecting the other family. oCRF and rCRF bind substantially similarly to both CRFR1 and CRFR2 families. A. Ruhmann et al. P.N.A.S., 95, 15264–15269 (December 1998) reported that [D-Phe11, His12]-sauvagine (11–40) was an antagonist that acted selectively with respect to CRFR2β and exhibited competitive antagonism equal to about 30% of that of the then best antagonist for CRFR1 and close to equal antagonism for CRFR2β compared to this previously best reported compound. Thereafter, the search has continued for CRF analogs that will serve as effective competitive antagonists to modulate the activation of CRFR2 while having even less effect upon CRFR1.